Hemopexin Is Synthesized in Peripheral Nerves but Not in Central ...

THEJOURNAL OF B I O L ~ I C CHEMISTRY AL 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 267, No. 15,Issue of May 25, pp. 1 ~ ~ 19921 Printed in U.S.A.

Hemopexin Is Synthesized in Peripheral Nerves but Not in Central Nervous System and Accumulatesafter Axotomy* (Received for publication, September 30,1991)

Jean-Paul SwertsSB, CathySoula$, YvesSagot$, Marie-Jose GuinaudyS, Jean-Claude Guillemotll, Pasquale Ferraraq, Anne-Marie DupratS, and Philippe CochardS From the $Centre de Biologie du Dckeloppement, Centre National dela Recherche Scientifique URA 675 affili6e a E’Imtitut N a t ~ ode ~ la l Santt; et de la Recherche M e d ~ a ~ , ~ n ~ uPaul e r s Sabatier, ite 31@2 Toulouse Cedez, and the %Unit6de ~ i o c h i ~ i e des Prott;ines, Sanofi Elf Bw-Recherches, B.P. 137,31328 LaiGge Cedex, France

In adult mammals, injured axons regrow over long axonal growth and extension after lesion remains to be demdistances in peripheral nerves but fail to do so in the onstrated for most of them. central nervous system.Analysis of molecular compoAnother approach in studying molecular determinants of nents of tissue environments that allow axonal re- axonal regeneration has been to analyze molecular changes growth revealed a dramatic increase in the level of accompanying Wallerian degeneration in peripheral nerves. hemopexin,a heme-transporting protein, in long-term This hasled to the identification of several soluble molecules axotomized peripheral nerve. In contrast, hemopexin that are specifically up-regulated after axotomy,including didnotaccumulateinlesionedoptic nerve. Sciatic nerve and skeletal muscle, but not brain,were shown nerve growth factor (17), glia-derived nexin (18), apolipoproto be sites of synthesis of hemopexin. Thus, hemopexin teins (19-21), and glial maturation factor @ (22). Again,most expression, which oan no longer be considered to be of these molecules are thought to play specific roles during liver-specific, correlateswith tissular permissivity for nerve repair. In this paper, we describe the strategy that led usto discover axonal regeneration. the presence of the plasma protein hemopexin in rat sciatic nerve and its considerable, long-lasting accumulation after nerve transection. In marked contrast, only low levels of the The success or failure of axonal regeneration in the adult protein were detected in brain and optic nerve, even after mammalian nervous system depends on interactions between lesion. Fu~hermore,we demonstrate that sites of hemopexin growing axons and their immediate microenvironment. For synthesis are notrestricted to liver parenchyma, as previously example, transplantation experiments (1, 2) have shownthat described (23) but are also present within the sciatic nerve neurons of both the PNS‘ and CNS can regenerate their and inskeletal muscle. Interestingly, no detectable hemopexin axons in the adult periphery, whereas they fail to do so in synthesis was found in the CNS. adult CNS tissue. Although it has become clear that glial cells EXPERIMENTAL PROCEDWRES and, to a lesser extent, other types of non-neuronal cells, are ~ a t e ~ ~ ~ bantiserum b i t directed against rat serum hemoinstrumental in controlling axon extension (for reviews, see pexin was kindly provided hy Pr. R. Engler and has been previously Refs. 3-8) the molecular determinants responsible for the characterized (24). Fluorescein isothiocyanate- and peroxidase-condifferenceinpermissivitybetween CNS and PNS tissues jugated anti-mouse and anti-rabbit IgG antibodies were from Nordic remain largely unknown. Immunology. Iscove’s modified Dulbecco’s medium was purchased In vitro assays using partially or totally purified molecules from Flow Laboratories. DEAE-Sepharose CL-GB was a product of have been invaluable in defining an increasingly large array Pharmacia LKB Biotechnology Inc. and Cm-Trysacryl M was from of factors r e ~ l a t i n gneuritic growth. Thus, two main classes IBF. Dimethyl p i m e l i ~ d a t e ,hemin-Agarose, and protein G-Sepha4 Fast Flow were obtained from Sigma. The C4 reversed-phase of components are thought to be responsible for these per- rose HPLC column was from Brownlee. [35S]Cysteine,[35S]methionine missive or nonpermissive effects: molecules allowingor pre- (specific activity > 1,000 Ci/mmol), Amplify, and Hyperfilm-MP venting growth cone anchorage and progression (4, 7, 9-13) were purchased from Amersham. All other chemicals wereof the and molecules acting as trophic or chemotatic factors (14- highest purity commercially available. Surgical Procedures-Adult Wistar rats (male or female, 200-300 16). However, the implication of these molecules in in situ

* This work wassupported by the Centre Nationalde la Recherche Scientifique and by grants from Association Francaise contre les Myopathies, MinistGre de la Recherche et de la Technologie, Centre National #Etudes Spatiales. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. § To whom correspondence should be addressed: Centre deBiologie du DQveloppement,Universitk Paul Sabatier,118route deNarbonne, 31062 Toulouse Cedex, France. Tel.: 33-61-55-64-23; Fax: 33-61-5565-07. ‘The abbreviations used are: PNS, peripheral nervous system; heme, iron-protoporphyrin IX; CNS, central nervous system; PBS, phosphate-buffered saline; DSNZab, DSNZ antibody; DSNZp, DSNZ protein;SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; HPLC, high-performance liquid chromatography.

g, IFFA CREDO) were anesthetized by intraperitonealinjection (0.5 mlfl00 g) of a chloral hydratesaline solution (chloral hydrate 8.5%, sodium chloride 8.5%). Nerve transections were performed unilaterally and intact nerves were used as controls. Sciatic nerve transection was as follows. The left sciatic nerve was exposed in the hip and transected between two ligatures. The distal stump was deflected on an adjacent muscle to prevent regeneration. The optic nerve transection was as follows. The posterior pole of the left eye was exposed through a superior intraorbital approach. Optic nerve was transected as close as possible to theeyeball. At various times, from 2 days to 3 months after surgery, rats were killed by an overdose of chloral hydrate. Degenerating and intact nerves were excised and desheathed. Brain and skeletal muscle were removed from intact animals. DSN2 Monoclonal Antibody-Adult rat sciatic nerves were transected as described above. Three weeks later the nerves were pooled and homogenized in PBS. Adult Balbfc mice wereimmunized against

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Hemopexin in Peripheral and Central Nervous System 0.5 ml of the homogenate emulsified in the samevolume of complete Freund's adjuvant. Mice were boosted 3 weeks later with the same homogenate in 50% incomplete Freund's adjuvant. A second boost was performed a month later, using crudehomogenate without adjuvant, followed by daylyinjectionsfor 3 days. On the fourth day, spleen cells were fused in polyethylene glycol 1500with SP2/0 myeloma cells and the cell suspension was plated in 24-well plates (Nunc). Culture and cloning of hybridomas were carried out according t o standard methods. For all experiments, DSN2ab was used as a culture supernatant of DSN2 hybridoma grown in Iscove's modified Dulbecco's medium supplemented with 10 pg/ml insulin and 20 p M ethanolamine. Immunohistochemistry-One week after lesion, nerveswere rapidly dissected andfixed with 4% paraformaldehyde in PBS.Fixed tissues were embedded for cryostat sectioning. Sections were incubated first with DSN2ab (culture supernatant, diluted1/10) or with rabbit antihemopexin antiserum (dilution, 1/2000) and, after extensive washing, with fluorescein isothiocyanate-conjugatedanti-mouse IgG (dilution, 1/100) or with fluorescein isothiocyanate-conjugatedanti-rabbit IgG (dilution, 1/50) antibodies, respectively. Sections were mounted in Moewiol and viewed under epifluorescence. Immunoblotting-Freshly dissected tissues were homogenized in 50 mM Tris-HCI, 130 mM sodium chloride, 2 mM EDTA, and 2 mM phenylmethylsulfonyl fluoride, at pH7.4. The crude homogenate was cleared (10,000 X g for 15 rnin), and total proteins (50pg)were separated by electrophoresis in a 10% gel, in the absenceof reducing agent. After wet electrophoretic transfer to a nitrocellulose membrane, proteins were analyzed with DSN2ab (dilution1/40) or rabbit anti-rat hemopexin antiserum (dilution 1/6400), and indirect immunoperoxidase staining. The intensityof immunological reactions was estimated on densitometric scans (Vernon scanner) performed rapidly after the gel was dried and rendered transparentby pretreating with Eukitt (Merck). Immunoaffinity Gel Preparation-Thisprocedure was adapted from the method of coupling antibodies on protein A beads previously described (see Ref. 25). One liter of supernatant of DSN2 hybridoma grown in defined medium was brought to 50% of ammonium sulfate and incubatedfor 6 ha t 4 "C withstirring. After centrifugation (3000 X g for 30 min), the pellet was solubilized,exhaustivelydialyzed against a 0.1 M sodium phosphate, 0.15 M sodium chloride buffer a t p H 8.0, and mixed for 1 h a t room temperature with 1 ml of protein G-Sepharose pre-equilibrated withthe samebuffer. Afteran extensive washing with 0.2 M sodium borate,DSN2ab was immobilized on protein G-Sepharose beadsby dimethyl pimelimidate (20 mM) for 30 min under gentle rocking. The coupling reaction was stopped by 0.2 M ethanolamine. DSN2 Protein Purification-Striated muscles were homogenized with an Ultraturax inbuffer A (10 mM sodium phosphate) at pH7.4. After centrifugation (10,000 X g for 15 min), the supernatant was recentrifuged a t 100,000 X g for 30 min. The cleared homogenate fraction was chromatographed on a DEAE-Sepharose column equilibrated with buffer A at pH6.0. DSN2p was eluted by 50 mM sodium chloride in the samebuffer. DSN2p-enriched fractionswere adjusted t o 100 mM sodium phosphate, 150 mM sodium chloride, p H 8.0, and passed through a protein G-Sepharose column and then through the DSN2ab immunoaffinity column (see above).DSN2p was eluted from the latter columnwith100 mM triethanolamine at pH 11.5 and immediately neutralized with acetic acid. All experiments were performed a t 4 "C. Serum Hemopexin Purification-Rat serum was purified by a sequence of three chromatographic procedures. Proteins retained on a Cm-Trisacryl column, equilibrated with 10 mM sodium citrate, p H 4.9, were eluted by a linear gradient of 10-100 mM sodium citrate, p H 4.9. Fractions containing hemopexin were applied to column of DEAE-Sepharose, equilibrated with 10 mM sodium phosphate, p H 6.2, and elutedwith 150 mM sodiumchloride in the same buffer. Finally,hemopexinwaspurified to homogeneity using a heminagarose affinity column (see Ref. 26). Amino Acid Sequencing-Purified DSN2p was either injected on a C4 reversed-phase HPLC column and eluted ina single peak after 20 min by a 10-20% gradient of acetonitrile in HzO, containing 0.1% trifluoroacetic acid, or eluted from an Immobilon membrane after Western blotting. The N-terminal sequence of DSN2p was determined on an Applied Biosystem Sequencer (470A) coupled to an Applied Biosystem phenylthiohydantoin amino acid analyzer (120A). Metabolic Labeling and Immunoprecipitation-Transected aad contralateral intact sciatic nerves were dissected 5-6 weeks after surgery. For comparisons of hemopexin synthesis in intact and in-

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jured nerves, care was taken to incubate nerve segments of comparable length. Muscle and brain were dissected from a nonoperated rat. Tissueswere choppedand incubated with 100 pCi of ["S]cysteine or [35S]methionine in1 ml of oxygenated Tyrode's solution for 6-8 h a t 37 "C. Incubation medium and tissues were either collected separately or pooled. Intracellular proteins were released by freeze-thawing of the tissue. After centrifugation (10,000 X g for 15 min), the pellet was discarded. Incubation media and cleared tissue homogenates were incubated with protein G-Sepharose beads and pelleted by a shortcentrifugation.Supernatant was incubatedfirst with DSN2aband secondwith proteinG-Sepharose beads to form a precipitable antigen-antibody-protein G-Sepharose beads complex. All incubations were performed a t 4 "C for 1 h. Beads were washed three times in PBS, pH8, and the complex was then dissociated by heating for 10minin gel application buffer. Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis as described above, except for some experiments performed in the presence of 8-mercaptoethanol. The gel was fixed, soaked in Amplify, dried, and visualized by autoradiography on Hyperfilm-MP. Levels of isotopically labeled hemopexin were estimated by scanning autoradiograms with an LKB UltroscanXL.

RESULTS

Inorder to analyze at the molecular level environments that are permissive for axonal regeneration, monoclonal antibodies were raised against lesioned sciatic nerve. Mice were immunized against whole extracts of the distal stump of rat sciatic nerves removed 3 weeks after transection. After fusion with myeloma cells, immunohistochemicalscreening was used to select those hybridomas secreting antibodies that recognized antigens present in lesioned PNS tissue but absent from intact or lesioned CNS tissue. Among several antibodies fulfilling the desired criteria, one, DSNPab, recognized an antigen (DSNPp) present in sections of intact sciatic nerve (Fig. lA ) and displayed much higherimmunoreactivity in the distal nerve stump 8 days after transection (Fig. 1B).Immunolabeling was restricted to the periphery of axonal units and of blood vessels in intact nerve. After lesion, immunoreactivity appeared morediffuse and in some instances was found within axonal units (Fig 123). In addition, strong immunoreactivity was noted at the periphery of myotubes in skeletal muscle (Fig. 1D).In contrast, DSN2p immunoreactivity was undetectable in sections of intact optic nerve and was expressed E

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FIG. 1. Immunolocalization of DNS2p in intact and denervated tissues (A-D) a n d its characterization in tissue extracts ( E ) . In control nerve (A) and muscle ( D ) ,DSN2p is localized in the periphery of axonunitsand of muscle fibers. Immunoreactivity appears more diffuse and is greatly enhanced in the distal stump of the transected sciaticnerve ( B ) ,whereas only weak staining is found in lesioned optic nerve ( C ) .Scale bars, 50 Fm (A-C) and 25 pm (D). E, Western blot analysis of the tissue distributionof DSN2p confirms the immunofluorescence data. Lane 1 , intact sciatic nerve; lune 2, transected sciaticnerve; lune 3, intact brain; lane 4, intact optic nerve; lane 5, transected optic nerve; lane 6, intact skeletal muscle.

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serum hemopexin (30);(iii) both proteins display an absorponly faintly 8 days after nerve transection (Fig. IC). The pattern of immunoreactivity in lesioned optic nerve is sugges- tion peak a t 413 nm (26). Furthermore, muscle DSN2p and heme affinity-purified rat serum hemopexin were both imtive of a localization on astrocytic processes (Fig. 1C). Immunoblots of soluble extracts of nerve and muscle (Fig. munostained on Western blots by DSN2ab (Fig. 3). A rabbit antiserum of high titer raised against rat hemopexin (24) 1E) revealed that, under nonreducing conditions,DSN2p migrated as a doublet, its major band being of an apparent recognized major bands of 57 (Fig.4C) and 69 kDa (not molecular mass of 57 kDa in all tissues considered. Under shown), under nonreducing and reducing conditions, respecreducing conditions, the epitope was lost,indicating that tively, after SDS-PAGE of extracts of intact and lesioned DSN2ab recognizes a conformational structure of the antigen sciatic nerve, thus confirming the identity of nerve DSN2p depending on one or more disulfide bonds. Quantification by with hemopexin. On both sections and immunoblots of intact scanning immunoblots confirmed the results of immunocy- and denervated sciatic nerve (Fig. 4)) thisantiserum yielded tochemical analysis. The increase inDSN2p in the distal immunoreactivities very similar to those displayed by nerve stump after permanent lesion was found to be long- DSN2ab. This demonstrates that theincreased DSN2p/hemlasting: 2 weeks after lesion, the relative amount of DSN2p opexin immunoreactivity observed after lesion is not merely was 7 to 10 times higher than in controls and thisaccumula- due to changes in thespecificity of DSNZab, possiblyresulting tion persisted for at least 3 months. In marked contrast, the from posttranslational modification of the protein, but rather protein was hardly detectable in brain and inoptic nerve and reflects an increase in nerve hemopexin content. Although hemopexin is believed to be a liver-specific prodincreased only slightly in the latter after transection (Fig. uct (23), the striking accumulation of the proteinin the 1E). Since DSN2ab recognizes a conformational epitope, we lesioned nerve and itspresence in muscle incited us todetercould not hope to clone the gene via an expression library. mine whether these tissues were capable of synthesizing it. We thereforetook advantage of the fact that DSN2p is present We therefore performed radioactive amino acid incorporain relatively large amounts in muscle to purify it from this tions insciatic nerve, skeletal muscle, and brain in uitro followedby immunopurification. Autoradiographic analysis tissue (see “Experimental Procedures”). Soluble proteins from muscle were firstseparatedon of immunoprecipitated proteins separated by SDS-PAGE reDEAE-Sepharose, yielding a 150-fold purified preparation of vealed that hemopexin was indeed synthesized within sciatic DSN2p. A nearly homogeneous antigen was then obtained 1 2 3 4 following an immunoaffinity purification step on protein-(=Sepharose coupled to DSN2ab (Fig. ?A).The sequence of the first 20 N-terminal amino acids of the purified antigen was identical to the N-terminal sequence of hemopexin from rat plasma (23,27) (Fig. 2B). That muscle-DSN2p is identical to hemopexin was confirmed by the following experiments. After partial hydrolysis by endoglycosidase-F (Genzyme) of the carbohydrate moiety of the purified protein, Western blot analysis conditions revealed several bands running between 57 and 46 kDa under unreduced conditions, thus indicating FIG. 3. Immunoblotting analysisof hemopexin from various that DSN2p is a glycoprotein with N-linked oligosaccharides sources, using DSN2ab. Hemopexin purified from rat serum by (data not shown), as is authentic rat hemopexin (28). More- heme affinity chromatography (lane I ) and from rat skeletal muscle over, DSN2p and rathemopexin display similar physicochem- by immunoaffinity (lane 2; see Fig. 2 A ) ; the amounts of deposited ical properties: (i) due to a compacted conformation main- protein were not determined. Total extracts (30 pg) from 3-weektained by disulfide bonds, their apparentmass shifts from 57 lesioned sciatic nerve (lane 3) and from control nerve (lane 4 ) . to 69 kDa when SDS-polyacrylamide gel electrophoresis is Electrophoresis was performed in the absence of reducing agent. performed under reducing conditions (29) (Fig. !?A).Although albumin displays an identical electrophoretic behavior (e.g. see Ref. 261,we have verified that DSN2ab does not crossreact to any extent with albumin; (ii)purified DSN2p migrates in two-dimensional gel electrophoresis as a series of spots of acidic pHi (data notshown), abehavior also characteristic of A

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FIG. 2. SDS-PAGE analysis ( A ) and sequence of 20 N-terminal amino acids ( B ) of purified DSN2p. A , silver-stained DSN2ab-immunopurified proteins (100 ng) migrate on SDS-PAGE as major bands of 57 and 69 kDa, respectively, under reducing (lane I ) or nonreducing (lane 2) conditions. The presence of a minor 57kDa band in lane I indicates incomplete reduction of DSN2p. B, comparisons of N-terminal amino acid sequences of DSN2p (upper line) and of rat serum hemopexin (from Refs. 23, 27) (lower line). The question marks indicate nonidentified amino acids.

of FIG. 4. Localizationbyindirectimmunofluorescence hemopexin in denervated and intact sciatic nerve, using antirat hemopexin antiserum.Transverse sections of the distal stump of the nerve 8 days after transection ( A ) and of the contralateral intact nerve ( B ) . For direct comparison, both sections havebeen photographed and printed with identical exposure times. Immunoreactivity is markedly enhanced in lesioned nerve as compared to intact nerve. Note that the distribution of the reaction product is similar to thatfound with DSN2ab (see Fig. 1).Scale bars, 50 pm. C, protein extracts (30 pg in each lane) from segments of the same lesioned (lanes 1 , 3 ) and intact (lanes 2, 4 ) nerves used in A and B have been subjected to Western blot analysis. Lanes 1, 2, anti-rat hemopexin antiserum; lanes 3, 4, DSN2ab. The antiserum is highly specific for hemopexin. Both antibodies demonstrate a similar rate of increase in hemopexin content in lesioned sciatic nerve.

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muscle, a recent study indicates that hemopexin mRNA could not be detected in this tissue (23). This discrepancy may indicate that hemopexin in muscle, and perhaps also in sciatic nerve, is actively translated from low levels of mRNA. The cell types responsible for this synthesis in nerve and muscle remain to be identified. The dramatic accumulation of hemopexin in the nerve after lesion, evidenced by immunohistochemical and Western blot techniques using two different and unrelated antibodies, raises the question of whether the protein originates entirely from local synthesis or partly from extrinsic sources. At least in rat, hemopexin is overexpressed by the liver during the acute phase (26). Thus, raised serum levels could contribute to the accumulation of hemopexin in lesionednerve.However, this isunlikely since the rise in serum hemopexin during the acute phase is short-lived (32), whereas that in the nerve lasts several months. Moreover, the level of hemopexin remained unchanged in the contralateral intact nerve during the entire period examined. The muscle DISCUSSION itself cannot be a source of nerve hemopexin, since hemopexin When lesioned, the adult PNS andimmature CNS, but not also increases dramatically in sciatic nerves transected at the adult CNS, constitute very efficient environments for the both ends? regrowth of injured axons. We have undertaken a study of After nerve lesion, the distal nerve stump undergoes Walmolecular determinants representative of lesioned PNS tissue lerian degeneration. Nerve fibers and myelin sheaths become with the purpose of characterizing factors essential for axonal phagocytozed by proliferating Schwann cells and 'macroregeneration. An antibody probe,DSNZab,was generated phages that invade the site of lesion (6). During this phase of against degenerating rat sciatic nerve and selected by virtue degeneration, a number of soluble proteins, including apoliof greatly enhanced immunoreactivity in the PNSafter lesion poproteins (19-21), nerve growthfactor (17), glial maturation and of low or undetectable reaction in theCNS. Immunoblot- factor p (22), and glia-derived nexin (18), originating from ting experiments confirmed our initial results, showing that Schwann cells, endoneuria1 fibroblasts, or macrophages, are DSN2p immunoreactivity increased only slightly in the le- overexpressed; after nerve regeneration, their expression desioned CNS (optic nerve), reaching the level found in intact creases toward normal levels. A specific role in nerve reconPNS, whereas DSN2p accumulated strikingly in the degen- struction has been proposed for all of these proteins. Measerating distal stump of the sciatic nerve. While the patternof urement of hemopexin content in the proximal stump of immunoreactivity displayed by DSN2ab is consistent with an transected sciatic nerves together with the results of prelimiextracellular localization of the antigen, its association with nary experiments involving crush-induced lesion of nerve the extracellular matrix and/or the plasma membrane of indicate that hemopexinlevelsalso return toward normal Schwann cells and other nonneuronal cells remains a possi- after axonal regeneration. This suggests that hemopexin bility at the present time. expression, like that of the soluble proteins listed above, is Purification and characterization of DSN2p by biochemical, specifically regulated during PNS lesion and repair. physicochemical, and immunological criteria led to the idenThe role of hemopexin in the process of nerve regeneration tification of muscle and nerve DSN2p as hemopexin. In remains to be established. However, it appears to regulate addition, amino acid incorporations in tissues demonstrate gene expression of, at least, heme- and iron-related proteins that sciatic nerve and muscle, but not brain, synthesize sig- (33,34). This is achievedthrough the internalization of heme nificant levels of this protein. These results may seem to via specific heme-hemopexin receptors (33, 34). Within the diverge from the commonly held notion that hemopexin is a cell, heme isrequired for, or regulates the function of various liver-specific product (23). However, to our knowledge, hem- proteins, and thus participates in cell metabolism(see Ref. 35 opexin synthesis has not been previously examined in the for a review). The fact that among others, it regulates the PNS. Although we found hemopexin synthesis in skeletal activities of guanylate cyclase (36), eIF-Pa (37), a factor regulating translation, andseveral transcriptional factors (3841) endows hemewith potential roles in cell growth, survival, or differentiation. Such effects have not beenfullydocumented outside hematopoietic cell lineages (see Refs.42,43). However, heme has been shown to stimulate in vitro differentiation of muscle cells (44) and to promote neurite outgrowth as well as neuron survival (45, 46). These data and the fact that after lesion hemopexin levels are raised in the PNS, but notin the nonregenerating CNS, are in favor of the hypothesis that hemopexin, through its heme moiety, could FIG. 5. Synthesis and secretion of hemopexin analyzed by incorporations of ['"S]methionine (Zane I ) and ['"Slcysteine play a hitherto unsuspected role in nerve repair.

nerve and muscle, whereas isotopically labeled hemopexin was not detected in brain in these experiments (Fig. 5). In immunoprecipitates, the major excreted radiolabeled protein ran at 69 kDa under reduced conditions and 57 kDa in nonreduced gels, thus confirming its identitywith hemopexin. In addition, when extracted from both supernatant and tissues, hemopexin synthesized in vitro was resolved in reduced gels into an additional, exclusively intracellular, molecular form of 49 kDa (Fig. 5). The 69-kDa protein corresponds to the fully glycosylated form of hemopexin, whereas the 49-kDa protein is likely to represent the nonglycosylated form (31). Scanning autoradiograms revealed a 3-fold increase in total hemopexin neosynthesizedin 5-week lesionednerve compared to contralateral intact nerve (Fig. 5). Therefore, nerve lesion stimulated not onlyhemopexin accumulation but also its synthesis.

(lanes 2-5) into tissues in vitro. Radioactive hemopexin is released by intact sciatic nerve into theculture medium and runs, under nonreducing conditions, as a 57-kDa major protein (lane 1 ). Synthesis of hemopexin is increased 3-fold in transected ( l a n e 2) compared to intact (lane 3) sciatic nerve. Hemopexin synthesis is also found in muscle (lane 5 ) but remains undetectable in brain ( l a n e 4 ) . Under reducing conditions, total neosynthesized hemopexin migrates as two major bands of 49 and 69 kDa. As in Fig. 2, the minor 57-kDa band in lanes 2-3 indicates incomplete reduction of the protein.

Acknowledgments-Initial phases of this work, in particular the production of monoclonal antibodies, were carried out while P. C. was at the Neuroscience Unit of the Montreal General Hospital Research Institute, McGill University, Montreal, Canada. We wish

* J.-P. Swerts, Y. Sagot, M.-J. Guinaudy, and P. Cochard, unpublished results.

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Hemopexin in Peripheral and Central Nervous System

to express our deep gratitude to A. Aguayo for generating new ideas and very stimulating discussions, supervising the early stages of this work, and commenting on initial versions of the manuscript. We are grateful to Pr. R. Engler for the generous gift of anti-hemopexin antiserum. We also thank J. Smith for very helpful and thorough critical review of the manuscript. REFERENCES 1. Aguayo, A., David, S., Richardson, P., and Bray, G. (1982) Adv. Cell Neurobwl. 3 , 215-234 2. Aguayo, A. J. (1985) in Synaptic Plasticity (Cotman, W., ed) pp. 457-488, Guilford, New York 3. Bunge, R. P., Kleitman, N., Ard, M. D., and Duncan, I. D. (1988) in Progress in Brain Research (Gash, D. M., and Sladek, J. R., Jr., eds) Vol. 78, pp. 321-326, Elsevier Science Publishers BV, Amsterdam 4. Reichardt, L. F. (1988) in Current Issues in Neural Regeneration Research, pp. 119-125, Alan R. Liss, Inc., New York 5. Smith, G. M., and Silver, J. (1988) in Progress in Brain Research (Gash, D. M., and Sladek, J. R., Jr., eds) Vol. 78, pp. 353-361, Elsevier Science Publishers BV, Amsterdam 6. Fawcett, J. W., and Keynes, R. J. (1990) Annu. Rev. Neurosci. 13,43-60 7. Schwab, M. E. (1990) Trends Neurosci. 13,452-456 8. Fawcett, J. W. (1991) Curr. Biol. 1,55-56 9. Carbonetto, S., Douville, P., Harvey, W., and Turner,D. C. (1988) in Current Issues in Neural Regeneration Research, pp. 147158, Alan R. Liss, Inc., New York 10. Jessell, T. M. (1988) Neuron 1,3-13 11. Patterson, P. H. (1988) Neuron 1,263-267 12. Sanes, J. R. (1989) Annu. Rev. Neurosci. 12,491-516 13. Lander, A. D. (1990) Curr. Opin. Cell Biol. 2,907-913 14. Levi-Montalcini, R., and Angeletti, P. U. (1968) Physiol. Rev.48, 534-569 15. Barde, Y.-A. (1989) Neuron 2, 1525-1534 16. Tessier-Lavigne, M., and Placzek, M. (1991) TrendsNeurosci. 14,303-310 17. Heumann, R., Korsching, S., Bandtlow, C., and Thoenen, H. (1987) J. Cell Biol. 1 0 4 , 1623-1631 18. Meier, R., Spreyer, P., Ortmann, R., Harel, A., and Monard, D. (1989) Nature 342,548-550 19. Muller, H. W., Gebicke-Hiirter, P. J., Hangen, D. H., andShooter, E. M. (1985) Science 2 2 8 , 499-501 20. Ignatius, M. J., Gebicke-Harter, P. J., Skene, J. H. P., Schilling, J. W., Weisgraber, K. H., Mahley, R. W., and Shooter, E. M. (1986) Proc. Natl. Acad. Sci. U.S. A . 8 3 , 1125-1129 21. Spreyer, P., Schaal, H., Kuhn, G., Rothe, T., Unterbeck, A., Olek, K., and Muller, H.W. (1990) EMBO J. 9,2479-2484 22. Bosch, E.P., Zhong, W., and Lim, R. (1989) J. Neurosci. 9,36903698

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